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Enhancing the function of CD34(+) cells by targeting plasminogen activator inhibitor-1.

Hazra S, Stepps V, Bhatwadekar AD, Caballero S, Boulton ME, Higgins PJ, Nikonova EV, Pepine CJ, Thut C, Finney EM, Stone DJ, Bartelmez SH, Grant MB - PLoS ONE (2013)

Bottom Line: Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications.To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO.We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34(+) cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34(+) cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34(+) cells. To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+) cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.

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Diabetic individuals protected from development of microvascular complications exhibit reduced expression of PAI-1 and increased expression of uPA in CD34+ cells.Microarrays were conducted on CD34+ cells obtained from diabetic individuals with microvascular complications (n=5), diabetic individuals without microvascular complications (n=5), healthy age-matched controls (n=4). mRNA transcript levels in cells from protected diabetic individuals were compared to those from diabetic individuals with complications by t-test. 270 probe sets were found differentially expressed with p<0.001 (false discovery rate ~19%). (a) Ingenuity pathway map shows the gene expression profile. In CD34+ cells from protected individuals, TGF-β1, TGF-βR1, TGF-βR2, PAI-1 and tPA (tissue plasminogen activator) were downregulated whereas uPA (urokinase plasmonigen activator) was upregulated. (b) Microarray data was confirmed by real-time RT-PCR showing that protected individuals have low levels of PAI-1 mRNA compared to diabetic individuals.
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pone-0079067-g001: Diabetic individuals protected from development of microvascular complications exhibit reduced expression of PAI-1 and increased expression of uPA in CD34+ cells.Microarrays were conducted on CD34+ cells obtained from diabetic individuals with microvascular complications (n=5), diabetic individuals without microvascular complications (n=5), healthy age-matched controls (n=4). mRNA transcript levels in cells from protected diabetic individuals were compared to those from diabetic individuals with complications by t-test. 270 probe sets were found differentially expressed with p<0.001 (false discovery rate ~19%). (a) Ingenuity pathway map shows the gene expression profile. In CD34+ cells from protected individuals, TGF-β1, TGF-βR1, TGF-βR2, PAI-1 and tPA (tissue plasminogen activator) were downregulated whereas uPA (urokinase plasmonigen activator) was upregulated. (b) Microarray data was confirmed by real-time RT-PCR showing that protected individuals have low levels of PAI-1 mRNA compared to diabetic individuals.

Mentions: We hypothesized that diabetic individuals protected from the development of microvascular complications might have more robust CD34+ cell function with a superior reparative response compared to CD34+ cells from diabetic individuals manifesting vascular complications. We identified a unique diabetic cohort without microvascular complications despite having diabetes for more than 40 years with largely poor metabolic control throughout this entire time. CD34+ cells from this cohort of protected subjects showed increased migratory potential compared to cells from diabetic subjects with microvascular complications [30]. Using gene array studies, we compared the CD34+ cells from protected diabetic individuals to diabetic individuals with microvascular complications that were matched for sex, age and glucose control, as well as to healthy controls using Affymetrix microarrays (Table 1). 270 probe sets were found differentially expressed with p<0.001 (false discovery rate ~19%) (Figure 1a). Ingenuity pathway map shows the gene expression profile. In CD34+ cells from the protected individuals, TGF-β1, TGF-βR1, TGF-βR2, PAI-1 and tPA (tissue plasminogen activator) were downregulated whereas uPA (urokinase plasmonigen activator) was upregulated. We also measured PAI-1 expression in these three cohorts by real-time RT-PCR, which showed that protected individuals had low levels of PAI-1 in CD34+ cells compared to diabetic individuals (Figure 1b). These results suggest that the “protected” subjects had reduced activation of the TGF-β1/PAI-1 system.


Enhancing the function of CD34(+) cells by targeting plasminogen activator inhibitor-1.

Hazra S, Stepps V, Bhatwadekar AD, Caballero S, Boulton ME, Higgins PJ, Nikonova EV, Pepine CJ, Thut C, Finney EM, Stone DJ, Bartelmez SH, Grant MB - PLoS ONE (2013)

Diabetic individuals protected from development of microvascular complications exhibit reduced expression of PAI-1 and increased expression of uPA in CD34+ cells.Microarrays were conducted on CD34+ cells obtained from diabetic individuals with microvascular complications (n=5), diabetic individuals without microvascular complications (n=5), healthy age-matched controls (n=4). mRNA transcript levels in cells from protected diabetic individuals were compared to those from diabetic individuals with complications by t-test. 270 probe sets were found differentially expressed with p<0.001 (false discovery rate ~19%). (a) Ingenuity pathway map shows the gene expression profile. In CD34+ cells from protected individuals, TGF-β1, TGF-βR1, TGF-βR2, PAI-1 and tPA (tissue plasminogen activator) were downregulated whereas uPA (urokinase plasmonigen activator) was upregulated. (b) Microarray data was confirmed by real-time RT-PCR showing that protected individuals have low levels of PAI-1 mRNA compared to diabetic individuals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815099&req=5

pone-0079067-g001: Diabetic individuals protected from development of microvascular complications exhibit reduced expression of PAI-1 and increased expression of uPA in CD34+ cells.Microarrays were conducted on CD34+ cells obtained from diabetic individuals with microvascular complications (n=5), diabetic individuals without microvascular complications (n=5), healthy age-matched controls (n=4). mRNA transcript levels in cells from protected diabetic individuals were compared to those from diabetic individuals with complications by t-test. 270 probe sets were found differentially expressed with p<0.001 (false discovery rate ~19%). (a) Ingenuity pathway map shows the gene expression profile. In CD34+ cells from protected individuals, TGF-β1, TGF-βR1, TGF-βR2, PAI-1 and tPA (tissue plasminogen activator) were downregulated whereas uPA (urokinase plasmonigen activator) was upregulated. (b) Microarray data was confirmed by real-time RT-PCR showing that protected individuals have low levels of PAI-1 mRNA compared to diabetic individuals.
Mentions: We hypothesized that diabetic individuals protected from the development of microvascular complications might have more robust CD34+ cell function with a superior reparative response compared to CD34+ cells from diabetic individuals manifesting vascular complications. We identified a unique diabetic cohort without microvascular complications despite having diabetes for more than 40 years with largely poor metabolic control throughout this entire time. CD34+ cells from this cohort of protected subjects showed increased migratory potential compared to cells from diabetic subjects with microvascular complications [30]. Using gene array studies, we compared the CD34+ cells from protected diabetic individuals to diabetic individuals with microvascular complications that were matched for sex, age and glucose control, as well as to healthy controls using Affymetrix microarrays (Table 1). 270 probe sets were found differentially expressed with p<0.001 (false discovery rate ~19%) (Figure 1a). Ingenuity pathway map shows the gene expression profile. In CD34+ cells from the protected individuals, TGF-β1, TGF-βR1, TGF-βR2, PAI-1 and tPA (tissue plasminogen activator) were downregulated whereas uPA (urokinase plasmonigen activator) was upregulated. We also measured PAI-1 expression in these three cohorts by real-time RT-PCR, which showed that protected individuals had low levels of PAI-1 in CD34+ cells compared to diabetic individuals (Figure 1b). These results suggest that the “protected” subjects had reduced activation of the TGF-β1/PAI-1 system.

Bottom Line: Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications.To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO.We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34(+) cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34(+) cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34(+) cells. To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+) cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.

Show MeSH
Related in: MedlinePlus